BR112014024968B1 - SYSTEMS IN THE FORM OF BIOIMPLANTS WITH MIXTURE OF PARTICULATE IN POLOXAMER GEL FOR MULTIPHASE RELEASE OF GROWTH FACTORS - Google Patents

Abstract

SYSTEM AND METHOD FOR MULTIPHASE RELEASE OF GROWTH FACTORS, AS WELL AS THE USE OF ITS COMPONENTS. A system for multiphased delivery of at least one growth factor to a treatment site comprises a delivery vehicle for delivering at least one growth factor in an initial release profile and a carrier for delivering at least one growth factor in an initial release profile. of sustained release. The initial release profile releases at least one growth factor over a period of hours to days, which growth factor is released in large amounts initially, with the rest being released in progressively lower amounts. The sustained-release profile releases at least one growth factor over a period of days to weeks, where the growth factor is released by a generally constant amount over that period. The system is particularly suited for bioimplant applications. The invention also comprises methods and kits for multiphasic delivery of at least one growth factor. The invention also comprises calcium sulfate as a vehicle to deliver at least one growth factor in single and multiphase systems to deliver at least one growth factor to one location (...).

Description

CROSS-REFERENCE TO PREVIOUS PATENT APPLICATIONS

[0001] This patent application is a continuation patent application in part of PCT/CA2012/050234, filed on April 11, 2012, which claims priority under the Paris Convention of US Patent Application 61/474,049, filed April 11, 2011, the entire contents of each patent application are incorporated into this application by reference.

FIELD OF THE INVENTION

[0002] This invention relates to systems and methods for releasing biological substances. In particular, the invention relates to the release of growth factors associated with bioimplants. More particularly, the invention provides a system and method for producing a multiphase release profile of at least one growth factor to improve bioimplant performance.

BACKGROUND OF THE INVENTION

[0003] Growth factors (GFs) are peptides and proteins that stimulate the growth and/or differentiation of cells via the interaction of GFs with specific cell surface receptors. Growth factors play an integral role in tissue repair and regeneration and exogenous application of GFs can be used to stimulate repair of various tissues and organs including bone, cartilage, skin and mucous membranes and to enhance tissue repair by stimulating the angiogenesis at the repair site.

[0004] The transforming growth factor beta (TGFβ) superfamily of secreted growth and differentiation factors in mammals has more than 30 members. These dimeric proteins are characterized by a conserved structure based on seven cystine knots. They regulate the proliferation, differentiation and migration of many cell types, and have important roles in morphogenesis, organogenesis, tissue maintenance and wound healing. The TGFβ superfamily of growth factors can be subdivided into several subfamilies including the transforming growth factor beta subfamily, bone morphogenetic protein (BMP), and growth and differentiation factor (GDF) family (also called the BMP subfamily). ), and inhibin and activin subfamily.

[0005] The BMP subfamily of the TGFβ superfamily comprises at least twenty proteins, including BMP-2, BMP-3 (also known as osteogenin), BMP-3b (also known as growth and differentiation factor 10, GDF-10) , BMP-4, BMP-5, BMP-6, BMP-7 (also known as osteogenic protein 1, OP-1), BMP-8 (also known as osteogenic protein 2, OP-2), BMP-9, BMP -10, BMP-11 (also known as growth and differentiation factor 8, GDF-8 or myostatin), BMP-12 (also known as growth and differentiation factor 7, GDF-7), BMP-13 (also known as growth and differentiation factor 6, GDF-6), BMP-14 (also known as growth and differentiation factor 5, GDF-5), and BMP-15 (for a review, see eg Azari et al. Expert Opin Invest Drugs 2001; 10:1677-1686).

[0006] BMPs have been shown to stimulate matrix synthesis in chondroblasts; stimulate alkaline phosphatase activity and collagen synthesis in osteoblasts, induce differentiation of early mesenchymal progenitors into osteogenic cells (osteoinduction), regulate chemotaxis of monocytes and mesenchymal cells, and regulate neural cell differentiation (for a review, see for example, Azari et al., Expert Opin Invest Drugs 2001;10:1677-1686 and Hoffman et al.Appl.Microbiol.Biotech 2001;57:294-308).

[0007] One of the many functions of BMP proteins is to induce the formation of cartilage, bone and connective tissue in vertebrates. The most osteoinductive members of the BMP subfamily are BMP-2, BMP-4, BMP-6, BMP-7, BMP-8 and BMP-9 (see, for example, Hoffman et al., Appl. Microbiol Biotech 2001, 57 -294-308; Yeh et al., J Cellular Biochem., 2005; 95-173-188; and Boden, Orthopedic Nursing 2005, 24:49-52). This osteoinductive ability of BMPs has long been considered very promising for a variety of therapeutic and clinical applications, including fracture repair; spine fusion; treatment of skeletal diseases, cranial regeneration, mandibular, and bone defects; and in oral and dental applications such as dentogenesis and cementogenesis during periodontal wound regeneration, extraction support grafting, alveolar crest augmentation, and sinus augmentation. Currently, recombinant human BMP-2 sold as INFUSE® by Medtronic FDA approved for use in spinal fusion surgery, for repair of fracture healing failures, and for use in oral surgery, while recombinant human BMP-7 sold as OP-1® by Stryker is approved as an alternative to autograft in the failure of recalcitrant consolidation of long bones and for revision posterolateral (intertransverse) lumbar spinal fusions, where autograft and bone marrow harvesting are not feasible or expected to promote Fusion.

[0008] Other recombinant growth factors that have been used exogenously to enhance bone repair include several TGFβs (see Clokie & Bell, J. Craniofacial Surg. 2003, 14:268-77), members of the fibroblast growth factor superfamily ( (s1 ):48-54), and vascular endothelial growth factor (VEGF) ( Street et al., 2002 PNAS 99:9656-61 ).

[0009] For these growth factors to be effective they must be active and available in a sufficient concentration at a time when critical densities of appropriate responder cells are present at the repair site. The short half-life, thermal instability, protease sensitivity and/or solubility of GFs require that their administration in combination with a carrier meet this requirement.

[00010] Several carriers have been evaluated for the delivery of GFs. These include fibrous collagen sponges, gelatin hydrogels, fibrin gels, heparin, reversed phase polymers such as poloxamers, carriers composed of polylactic acid (PLA), polyglycolic acid (PGA) or their copolymers (PLGA), PLGA carriers conjugated with heparin and inorganic materials such as calcium phosphates. For example, the bioimplant (GEM-21S®), which is used for periodontal regeneration, uses beta tricalcium phosphate (β-TCP) as the rhPDGF-BB carrier (see <http://www.osteohealth.com/GEM21S .aspx>).

[00011] However, these carriers are of limited effectiveness, due to loss of growth factor activity when associated with the carrier, inefficient release of GF at the implantation site, and/or poor protection against proteolysis and degradation. For example, the Infuse® bioimplant uses sponge type I collagen as the rhBMP-2 carrier. rhBMP-2 is released in a carrier burst and the half-life of BMP within the wound site is 1-3 days (Winn et al., 1998, Adv. Drug Del. Rev. 31:303; Friess e.g. al., 1999, Intl. J. Pharm., 187:91). So far, the mesenchymal stem cells, which regenerate bone, have migrated to the wound site in only fractions of one percent of the original amount of loaded BMP and are present to stimulate these cells to produce bone. The current solution to ensuring an effective level of BMP remaining at these later times is to significantly increase the amount of BMP that is initially loaded. These increased doses increase the risk of complications including bone formation beyond the implant site, immune autoresponders and potentially cancer. This still dramatically increases the cost of the implant.

[00012] Therefore, a need exists in the art for materials and methods that release growth factors with a profile that minimizes the amount of growth factor that must be loaded to achieve the required therapeutic effect.

[00013] One strategy is to encapsulate the GF in a biodegradable polymeric matrix that releases the GF with a sustained release profile over many days. For example, BMPs have been combined with polylactic acid (PLA) or polylactic coglycolic acid (PLGA) to produce sustained release profiles. However, incorporation of BMP into PLA or PLGA can denature BMP, reducing its activity, and it is difficult to manipulate the release profile to optimize bioimplant efficacy. Furthermore, the rate of degradation of these carriers is typically such that large amounts of GF remain trapped after curing is complete. Consequently, large amounts of GF have to be loaded into these polymers to ensure that sufficient GF is present at the appropriate times.

[00014] Another strategy is to chemically immobilize the GF directly on the surface of the vehicle. However, this can result in partial or complete loss of GF activity and can restrict GF activity such that only those cells directly in contact with the vehicle are able to interact with GF and respond (see Steinmuller-Nethl, D. et al. ., Biomaterials, 2006, 27: 4547-56) which may be undesirable as the effect may be limited to the immediate interface with the carrier and not along the wound site.

[00015] The composition of the carrier can influence the delivery of the GF. Calcium sulfate has been considered desirable as a bone substitute and GF carrier because it is osteoconductive, biodegradable, biocompatible and non-toxic (Chen et al., J. Craniofacial Surg., 2010, 21:188-197). However, it is also known that calcium sulfate has a rapid degradation rate when added to bone in situ and poor osteoinductive ability, which has limited its usefulness in bone implants.

[00016] One strategy for manipulating calcium sulfate degradation in situ was to control the rate of degradation by altering crystalline structures and adding polymers (eg, chitosan) to the calcium sulfate implant mixture (Chen et al., supra) . Polymer-coated calcium sulfate pellets that have been impregnated with BMP can reduce the rate of calcium sulfate resorption and increase the compressive strength and osteoinduction of the mixture (Chen et al., supra).

[00017] Composites containing hydroxyapatite (HAp), a major mineral component of bone, and calcium sulfate hemihydrate (CSH, gypsum) have been used in orthopedic grafts (eg, Damien, C et al., J. Biomed. Mat. Res., 1990, 24: 639-654; Damien, C et al., Spine, 2002, 16: S50-S58; Parsons, J., et al., Annals N.Y. Acad. Sci.). When CSH is mixed with sterile saline or water it immediately begins to gel. While in gel-state HAp, growth factors and/or various matrix components can be mixed together with the CSH to form the composite graft, which can then be inserted or injected into a bone defect where it establishes in situ. In such methods, CSH initially acts as a binder. However, subsequent resorption of calcium sulfate leaves a porous matrix behind with the bone growth space, which can be stimulated by growth factors in the hardened composite. Similarly, compositions for delivering osteogenic proteins including CSH, a porous particulate polymer blend, and an osteogenic protein are known (U.S. Patent 5,385,887 and U.S. Patent Application Publication No. 2008/0233165, each of which is incorporated into this application by reference as if presented in its entirety). In each of these methods, degradation of calcium sulfate is required for growth factor release. Therefore, bone regeneration is dependent on the rate of calcium sulfate degradation.

[00018] Bone grafts containing particulate bone and a biocompatible solid component comprising CSH and a calcium phosphate product are known, but do not involve the use of CSH or calcium phosphate as a growth factor carrier (Application Publication of U.S. Patent No. 2011/0208305, incorporated into this application by reference as if set forth in its entirety).

[00019] In nature, during wound healing, multiple GFs are present within the wound site and surrounding tissue in varying concentrations at different times. For example, immediately following a bone fracture, platelets at the site of injury will initially release large amounts of PDGF, with a sharp decline in protein levels within the fracture site over the next few days (see Tyndall et al., Clinical Orthopedics and Related Research , 2003, 408: 319-330). Conversely, BMP-2 is expressed at all stages of the fracture healing process (see Rasubala et al. British Journal of Oral and Maxillofacial Surgery, 2003, 41: 173178), although the amount of BMP-2 varies over time. of time (see Meyer et al. J Bone Jt. Surg 2003, 85-A: 1243-1254). The concentration of these growth factors is estimated to be orders of magnitude lower than those used during therapeutic application of exogenous GF due to the combination of concentration with cellular requirements and the synergistic effects of multiple growth factors. Producing a system that allows the delivery of growth factors with multiphase release profiles and the release of multiple growth factors with different release profiles would allow the use of bioimplants with GF release profiles that more closely mimic GF release during the process. of natural healing than current bioimplants that release a single growth factor in a burst or with sustained release.

[00020] This reference information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should it be construed, that any part of the foregoing information constitutes prior art against the present invention.

SUMMARY OF THE INVENTION

[00021] The present invention provides, in one aspect, a system, method and kit for the multiphasic release of at least one growth factor, for example, at a treatment site. For this purpose, the system of the invention can be provided as a bioimplant or the like. In one aspect, the method of the invention delivers at least one growth factor in an initial release followed by delivery of at least one growth factor in a "sustained release profile". The invention uses an initial release delivery system and a sustained release carrier.

[00022] In one aspect, the same growth factor is released in the initial and sustained release profiles. In another aspect, different growth factors are released, with a first growth factor released in an early profile and a second growth factor released in a sustained release profile. As will be known to those skilled in the art, it is believed that the release of two different growth factors in such different ways more closely mimics the natural growth factor delivery system at a treatment site.

[00023] As an aspect of the invention, there is provided a carrier that provides a sustained release of at least one growth factor, combined with a delivery vehicle that provides an initial release of at least one growth factor. The combination of carrier and delivery vehicle results in a multiphase release profile of the growth factor(s). In preferred embodiments, the amount of carrier and delivery vehicle is varied to control the release of at least one GF, wherein the amount of delivery vehicle and carrier is provided in a ratio of approximately 0.5 to 4.0: 1 (v:v). In preferred embodiments, the amount of delivery vehicle used is between 0.5 and 10.0 mL. In particularly preferred embodiments, 0.75-2.5 ml of delivery vehicle are used with 1 cm 3 of carrier. In particularly preferred embodiments, 1.0 mL of the delivery vehicle and 0.5 cm 3 of the carrier are used.

[00024] In preferred embodiments, the growth factor ("GF") is a member of the transforming growth factor beta (TGFβ) superfamily. In particularly preferred embodiments, the growth factor is a bone morphogenetic protein (BMP).

[00025] In one aspect of the present invention, the carrier ("CAR") is formed from calcium phosphate particles with a size of less than 80 microns and preferably less than 45 microns dispersed within a polymeric matrix that results in a larger structure. In one aspect, the structure is further coated with a layer of hydroxyapatite.

[00026] In one embodiment, at least one GF is applied as a liquid to the calcium particles and then lyophilized to the particles before combining with the polymer matrix. In some embodiments, 100% of the lyophilized GF associates with the particles. In other embodiments, less than 100% of the lyophilized GF associates with the particles and the remainder does not associate with the particles. Such a composition comprising GF-associated particles and lyophilized GF that does not associate with particles can be combined with a delivery vehicle such that the unassociated particles are distributed in the delivery vehicle, where they can be later released.

[00027] In another aspect of the present invention, the carrier is formed by mixing one or more calcium phosphate powders with a liquid solution containing at least one growth factor to produce a calcium phosphate cement. In one aspect, the cement is then ground into particles having a diameter of at least 100 microns and preferably between 0.3 and 3 mm in diameter.

[00028] In another aspect of the present invention, the carrier comprises particles of one or more calcium salts all with a diameter of at least 100 microns and preferably between 0.3 and 3 mm. A growth factor is then lyophilized onto the surface of the carrier particles.

[00029] In preferred embodiments, the delivery vehicle is a reversed phase polymer. In particularly preferred embodiments, the reversed-phase polymer is a poloxamer, more particularly poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) poloxamer 407 (also called Pluronic™ F127) at a concentration of at least 12% and preferably between 20 and 40%. In some particularly preferred embodiments, the amounts of P407 and carrier are varied to influence the amount of GF released from the carrier and optionally the delivery vehicle.

[00030] As indicated above, in one aspect, the carrier and delivery vehicle release the same growth factor while in another aspect, the carrier and delivery vehicle release different growth factors. In yet another aspect of the invention, the carrier and delivery vehicle are each adapted to deliver combinations of two or more growth factors, with the combination released by each being the same or different.

[00031] Thus, in one aspect, the invention provides a system for the multiphasic release of growth factors at a treatment site, the system comprising: - a delivery vehicle comprising at least a first growth factor; and - a carrier comprising at least a second growth factor; - wherein: - the delivery vehicle is adapted to release at least a first growth factor in an initial release profile over a first period of time; - the carrier is adapted to release at least a second growth factor in a sustained release profile over a second period of time.

[00032] In another aspect, the invention provides a method of multiphasic release of growth factors, the method comprising: - delivery of at least a first growth factor with an initial release profile; - delivery of at least a second growth factor in a sustained release profile.

[00033] In a further aspect, the invention provides a kit for the multiphase delivery of growth factors, the kit comprising: 1) - a delivery vehicle component; 2) - at least one first growth factor associated with the delivery vehicle; 3) - a carrier component; and 4) - at least one second growth factor associated with the carrier.

[00034] In yet a further aspect, the invention provides a kit for the multiphase delivery of growth factors, the kit comprising: - a delivery vehicle component; - a carrier component; - at least one first growth factor that is not associated with the delivery vehicle or carrier; and - at least one second growth factor associated with the carrier.

[00035] In one embodiment, the kit comprises at least two containers, wherein the first container comprises the delivery vehicle and the second container comprises the carrier associated with at least one second growth factor and at least one first growth factor. In preferred embodiments, at least a first growth factor is mixed with the delivery vehicle when the delivery vehicle is added to the carrier.

[00036] The present invention also provides, in one aspect, a system, method and kit for the release of at least one growth factor, for example, at a treatment site, in which the sulfate "carrier" particles calcium harbor at least one growth factor on their surface. For this purpose, the system of the invention can be provided as a bioimplant or the like.

[00037] In one aspect, the method of the invention delivers at least one growth factor in a "sustained release profile".

[00038] In one aspect of the present invention, the carrier comprises a mixture of calcium sulfate dihydrate and calcium phosphate particles. In preferred embodiments, the ratio of calcium sulfate to calcium phosphate particles is approximately 1:1 or 2:1.

[00039] In some aspects of the present invention, at least one growth factor is released in a single phase from the calcium carrier. In this aspect, GF is not released by the delivery vehicle. In other aspects, at least one growth factor undergoes multiphasic release from the calcium carrier and delivery vehicle. In preferred embodiments, the amount of carrier and delivery vehicle is varied to control the release of at least one GF, wherein the amount of delivery vehicle and carrier is provided in a ratio of approximately 0.5-4:1 ( v:v). In preferred embodiments, the amount of delivery vehicle used is between 0.5 and 10.0 mL. In particularly preferred embodiments, 0.5-2.5 ml of delivery vehicle is used per cm 3 of carrier. In particularly preferred embodiments, 1.0 mL of the delivery vehicle and 0.5 cm 3 of the carrier are used.

[00040] In one embodiment, at least one GF is applied as a liquid to the calcium particles and then lyophilized to the particles before combining with the polymer matrix.

[00041] In one embodiment, the GF is freeze-dried such that some GF associates with the carrier and some GF is separated from the carrier. When the delivery vehicle is added to the carrier, the separate GF becomes associated with the delivery vehicle.

[00042] In preferred embodiments, the distribution of at least one GF to the calcium particles is altered by varying the volume of the solution containing the GF in relation to the protein to be lyophilized to the particles. The amount of GF bound in calcium particles can be made higher by reducing the volume of solution used to deliver the GF. In preferred embodiments, lyophilization of GF to carrier particles is performed in a 1:1:0.5 ratio, where 1 unit of GF is mixed with 1 unit of the solution and lyophilized to 0.4 units of carrier. In particularly preferred embodiments, approximately 1.0 mg of GF is added to approximately 1.0 mL of the lyophilization solution for approximately 0.5 cm 3 of calcium particles.

[00043] In a further aspect the invention provides a kit for the delivery of growth factors, the kit comprising: - a delivery vehicle component; - a carrier component comprising a plurality of calcium sulfate particles; - at least one second growth factor associated with the carrier and optionally, - at least one first growth factor not associated with the carrier which will become associated with the delivery vehicle when the delivery vehicle is mixed with at least one first growth factor growth

[00044] In preferred embodiments, the carrier component of the kit further comprises calcium phosphate particles. In a particularly preferred embodiment, the ratio of calcium sulfate to calcium phosphate particles is approximately 1:1 or 2:1.

[00045] In a further aspect, the invention provides a kit for the multiphase delivery of growth factors, the kit comprising: - a delivery vehicle component; - at least one first growth factor associated with the delivery vehicle; - a carrier component comprising a plurality of calcium sulfate particles; and - at least one second growth factor associated with the carrier.

[00046] In preferred embodiments, the carrier component of the kit further comprises calcium phosphate particles. In a particularly preferred embodiment, the ratio of calcium sulfate to calcium phosphate particles is approximately 1:1 or 2:1.

BRIEF DESCRIPTION OF THE DRAWINGS

[00047] The invention will now be described with reference to the appended figures, which are briefly described below.

[00048] Figure 1 illustrates a sustained release profile exhibited by the carrier of the invention.

[00049] Figure 2 illustrates the initial release profile displayed by the delivery vehicle of the invention.

[00050] Figure 3 illustrates different release profiles based on an amount of growth factor in the delivery vehicle and carrier. A multiphase release profile is observed when growth factors are incorporated into both the delivery vehicle and the carrier (50-50).

[00051] Figure 4 illustrates the in vivo activity of bioimplants where a growth factor is released as shown in Figure 3 according to the method of the invention.

[00052] Figure 5 illustrates the formation of new bone (bone) in particles of calcium phosphate (CaP) when a bioimplant produced according to the method of the invention was implanted in a mouse.

[00053] Figure 6 illustrates the histological appearance of new bone (Bone) formed in a carrier (Carrier) when the bioimplant produced according to the method of the invention was implanted in a mouse.

[00054] Figure 7 illustrates a release profile of short stable growth factor produced by a carrier produced according to the method of the invention.

[00055] Figure 8 illustrates how a sustained release profile can be changed by modifying the properties of the carrier produced according to the method of the invention.

[00056] Figure 9A-D illustrates the lyophilized carrier, in which the volume of the lyophilized solution was varied, but the total lyophilized protein was fixed. Treatment groups 1 (A), 3 (B) and 5 (C) are represented.

[00057] Figure 10 A-B illustrates the histological appearance of new bone formed around the carrier components calcium phosphate (A) and calcium sulfate (B) used in a bioimplant produced according to the method of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[00058] Growth factors (GF) play an integral role in tissue repair and regeneration and exogenous GFs can be used to stimulate repair of various tissues and organs. For exogenous growth factors to be effective in stimulating repair, they must be conserved at the site requiring repair and protected from inactivation, sequestration, or degradation. To achieve this, carriers are used. However, the release of growth factors from known carriers is not ideal and cannot be easily modified. The current invention is based on: i) the finding that the multiphasic release of growth factors from a bioimplant increases the effectiveness of the implant; and ii) the finding that the use of calcium sulfate as a growth factor carrier can improve the potency of bioimplants containing GF.

[00059] The present inventors have developed methods and materials to increase the effectiveness, for example, of bioimplants by improving the release kinetics or release profile of growth factors at implantation sites, while maintaining the activity of the growth factors. In one aspect, the present invention provides a growth factor delivery system and method comprising a carrier that contains at least one growth factor, combined with a delivery vehicle that also contains at least one growth factor. At least one growth factor released by the carrier and delivery vehicle may be the same or different.

[00060] In another aspect, the present invention provides a growth factor delivery system and method that have increased effectiveness due to the use of a carrier comprising a plurality of calcium sulfate dihydrate particles comprising at least one GF in the its surface, combined with a delivery vehicle that may optionally contain at least one GF. In contrast, previous attempts to use calcium sulfate as a GF carrier have involved calcium sulfate particles that become incorporated or impregnated with the growth factor rather than coating the surface of the calcium particles with a GF. In the present invention, calcium sulfate degradation is not required for GF release.

[00061] In preferred embodiments, the amount of carrier and delivery vehicle is varied to control the release of at least one GF, in which the amount of delivery vehicle and carrier is provided in a ratio of approximately 0.5-4 0:1 (v:v). In preferred embodiments, the amount of delivery vehicle used is between 0.5 and 10.0 mL. In particularly preferred embodiments, 0.5-2.5 ml of delivery vehicle is used per cm 3 of carrier. In particularly preferred embodiments, 1.0 mL of the delivery vehicle and 0.5 cm 3 of the carrier are used.

[00062] In one embodiment, at least one GF is applied as a liquid to small calcium particles (<80 microns) and then freeze-dried to the particles before combining with a polymer matrix to produce the carrier structure.

[00063] In another embodiment, the GF is applied as a liquid to large particles (>100 microns) and then lyophilized with the particles resulting in a particle-associated and particle-free GF distribution.

[00064] In preferred embodiments, the distribution of at least one GF between being associated with the particles and being separated or "free" from the particles is altered by varying the volume of the solution containing the GF in relation to the amount of particles with which it is incubated before lyophilization. The amount of GF bound into particles can be made higher by reducing the volume of solution used to deliver the GF. In preferred embodiments, lyophilization of GF to carrier particles is performed in a 1:1:0.5 ratio, where 1 unit of GF is mixed with 1 unit of the solution and lyophilized to 4 units of carrier. In particularly preferred embodiments, approximately 1.0 mg of GF is added to approximately 1.0 mL of the lyophilization solution for approximately 0.5 cm 3 of calcium particles.

[00065] The system and method of the invention can be used for a variety of therapeutic and clinical applications, including: fracture repair; bone grafts; spinal fusion; and regeneration of cranial, mandibular and bone defects. For such applications, the system of the invention is preferably provided on or in the form of a bioimplant.

Definitions

[00066] Unless otherwise defined below, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[00067] As used in this application, the term "bioimplant" refers to a material that is suitable for implantation and contains an exogenous or biologically active growth factor. As discussed further in this application, the system of the present invention is preferably used by applying it to a bioimplant. The bioimplant is then delivered into an individual's body where the system releases at least one growth factor in a multiphase release profile.

[00068] As used in this application, the term "growth factor" refers to peptides and proteins that stimulate the growth and/or differentiation of cells via the interaction of GFs with specific cell surface receptors. Examples of growth factors include bone morphogenetic proteins (BMPs), transforming growth factor beta (TGFβ), insulin-like growth factors (IGF), fibroblast growth factors (FGFs), platelet-derived growth factor (PDGF ) and vascular endothelial growth factor. In preferred embodiments, the growth factors are BMPs.

[00069] By "recombinant" is meant a protein produced by a transiently transfected, stably transfected, or transgenic host cell or animal as directed by an expression construct containing the cDNA of that protein. The term "recombinant" also encompasses pharmaceutically acceptable salts of such polypeptide.

[00070] As used in this application, the term "polypeptide" or "protein" refers to a polymer of amino acid monomers that are alpha amino acids joined by amide bonds. Polypeptides, therefore, are at least two amino acid residues in length and are usually longer. Generally, the term "peptide" refers to a polypeptide that is only a few amino acid residues in length. A polypeptide, in contrast to a peptide, can comprise any number of amino acid residues. Therefore, the term polypeptide included peptides as well as longer sequences of amino acids.

[00071] As used in this application, the terms "bone morphogenetic protein" or "bone morphogenetic protein" or "BMP" are used interchangeably and refer to any member of the bone morphogenetic protein (BMP) subfamily of the bone-transforming factor superfamily growth factor beta (TGFβ) of growth and differentiation factors, including BMP-2, BMP-3 (also known as osteogenin), BMP-3b (also known as growth and differentiation factor 10, GDF-10), BMP- 4, BMP-5, BMP-6, BMP-7 (also known as osteogenic protein 1, OP-1), BMP-8 (also known as osteogenic protein 2, OP-2), BMP-9, BMP-10, BMP-11 (also known as growth and differentiation factor 8, GDF-8 or myostatin), BMP-12 (also known as growth and differentiation factor 7, GDF-7), BMP-13 (also known as and differentiation factor 6, GDF-6), BMP-14 (also known as growth and differentiation factor 5, GDF-5), and BMP-15.

[00072] The terms "bone morphogenetic protein" and "BMP" also encompass allelic variants of BMPs, conservative function variants of BMPs, and mutant BMPs that retain BMP activity. The BMP activity of such variants and mutants can be confirmed by any of the methods well known in the art (see section Assays for measuring BMP activity, below) or as described in Example 1

[00073] In preferred embodiments, the BMP is BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8 or BMP-9. In particularly preferred embodiments, the BMP is BMP-2, BMP-4 or BMP-7.

[00074] In preferred embodiments, the BMP is a mammalian BMP (e.g., mammalian BMP-2 or mammalian BMP-7). In particularly preferred embodiments, the BMP is a human BMP (hBMP) (e.g., hBMP-2 or hBMP-7).

[00075] As used in this application, the term "scaffold" refers to a material whose purpose is to provide a structure that supports cell adhesion, migration, and growth at a tissue repair site.

[00076] As used in this application, the term "carrier" refers to single or multiple components comprising materials and is adapted to release at least one growth factor at a treatment site in a "sustained release" profile during the period of time. In one aspect, the period of time taken by the carrier to release at least one growth factor is between several days and several weeks. Preferably, the carrier is adapted to release at least one growth factor over a period of weeks.

[00077] In preferred embodiments, the carrier also acts as a scaffold or matrix. As discussed above, in one aspect of the invention, the carrier is formed of calcium phosphate particles dispersed within a macroporous polymer framework or matrix. In one aspect, the scaffold or matrix is further coated with a layer of hydroxyapatite. In another aspect of the invention, the carrier is formed from calcium sulfate particles. In yet another aspect of the invention, the carrier is a mixture of calcium phosphate and calcium sulfate particles. In one embodiment, at least one growth factor is applied as a liquid to the calcium particles and then lyophilized to the particles before combining the particles with the polymer matrix. In preferred embodiments, the carrier is a solid.

[00078] As used in this order, the term "delivery vehicle" refers to a material that serves to transport the carrier. In one aspect of the invention, the delivery vehicle comprises or is associated with at least one growth factor and is adapted to deliver the at least one growth factor to a treatment site in an initial release profile over a period of time. . In other respects, the delivery vehicle does not initially comprise a growth factor. Rather, it is later combined with a GF that is not associated with the carrier prior to use, thereby producing the initial phase of GF release. In one aspect, the length of time taken by the delivery vehicle to release at least one growth factor is between several hours and several days. In a preferred embodiment of the invention, the delivery vehicle releases the majority of at least one growth factor in an "initial release" or "initial release profile" that lasts for a period of hours. Preferably, the delivery vehicle is adapted to release at least 80% of the growth factor(s) contained therein (or associated therewith) within a period of 72 hours. In preferred embodiments, the delivery vehicle is a liquid or a gel.

[00079] In one aspect, the delivery vehicle of the present invention can be used to facilitate the handling of carrier particles, wherein the combination of carrier and delivery vehicle results in the formation of a gel or bitumen.

[00080] In one aspect, the material that forms the delivery vehicle is in the form of a gel. In preferred embodiments, the delivery vehicle is a reversed phase polymer. As used in this application, the term "reversed phase" refers to the property whereby the polymer undergoes a reversible temperature-dependent transition from a liquid to a gel. In one aspect, the transition temperature is between 15°C and 37°C. Preferably, the transition temperature is between 15°C and 25°C. As would be known to those skilled in the art, "normal phase" materials increase in viscosity with a decline in temperature. In contrast, reversed-phase materials show a decline in viscosity when the temperature drops below their transition temperature.

[00081] In particularly preferred embodiments, the reversed phase polymer is a poloxamer, more particularly Pluronic™ F127 (also known as poloxamer 407 or P407).

[00082] In particularly preferred embodiments, the P407 polymer solution is between 20 and 40%

[00083] In preferred embodiments, the amount of carrier and delivery vehicles used in a bioimplant is altered to influence the amount of GF released from the bioimplant.

[00084] As used in this application, the term "sustained release" or "sustained release profile" refers to the release of at least one growth factor by the carrier over a period of several days or weeks with the amount released during a initial period that is similar to or less than the amount released during the same period several days or weeks after implantation. Preferably, a sustained release profile lasts at least one week. As will be appreciated by those skilled in the art, typically, the amount of growth factor released in a sustained release profile during the first three days will be less than the amount released during the next seven days.

[00085] As used in this application the term "initial release" or "initial release profile" refers to the initial release, by the delivery vehicle, of a large amount of at least one growth factor followed by progressively smaller amounts released during the period of hours or days. In one aspect, an initial release profile results in the delivery of at least 80% of the loaded growth factor(s) within a period of approximately 72 hours. An initial release profile is illustrated in Figure 2.

[00086] As used in this application, the term "multiphased release" refers to an initial release of at least one growth factor over an initial period of time, followed by "sustained" release of at least one growth factor over the course of a second period of time. Preferably, the initial period of time is approximately several hours and the second period of time is approximately several days to weeks. Such a release profile can also be referred to as "biphasic release" as it occurs in two stages. In preferred embodiments, the initial release is provided by the delivery system of the invention and the "sustained" release is provided by the carrier of the invention.

[00087] As used in this application, the term "potency" refers to a measure of drug activity expressed in relation to the amount required to produce an effect of given intensity.

[00088] In one aspect of the invention, the delivery vehicle component comprises at least 10% and not more than 50% of the total amount of growth factor(s) delivered by the system of the invention and the carrier component comprises at least 50% of the total amount of growth factor(s) delivered by the system.

Assays to measure BMP activity

[00089] Assays to characterize the in vitro and in vivo function of recombinant BMPs are well known in the art, (see, for example, U.S. Patent No. 4,761,471; U.S. Patent No. 4,789,732; U.S. Patent No. 4,795 804; U.S. Patent No. 4,877,864; U.S. Patent No. 5,013,649; U.S. Patent No. 5,166,058; U.S. Patent No. 5,618,924; U.S. Patent No. 5,631,142; U.S. Patent No. 6,150,328 ; U.S. Patent No. 6,593,109; Clokie and Urist, Plast.Restr.Surgi.2000;105:628-637;Kirsch et al., EMBO J 2000;19:3314-3324;Vallejo et al., J. Biotech 2002;94:185-194;Peel et al., J. Craniofacial.Surg.2003;14:284-291;and Hu et al., Growth Factors, 2004;22:29-33).

[00090] Such assays include: in vivo assays to quantify the osteoinductive activity of a BMP after implantation (e.g. in back muscle or thoracic area) in a rodent (e.g. a rat or a mouse) (see e.g. , U.S. Patent No. 4,761,471; U.S. Patent No. 4,789,732; U.S. Patent No. 4,795,804; U.S. Patent No. 4,877,864; U.S. Patent No. 5,013,649; U.S. Patent No. 5,166,058; U.S. Patent No. 5,618,924; U.S. Patent No. 5,631,142; U.S. Patent No. 6,150,328; U.S. Patent No. 6,503,109; Kawai and Urist., Clin. Orthop.Report. Res., 1988; 222: 262-267, Clokie and Urist, Plast.Restr.Surg., 2000;105:628-637;and Hu et al., Growth Factors, 2004;22:29-33); in vivo assays to quantify the activity of a BMP to regenerate trephinator-caused cranial defects in mammals (e.g., rats, dogs, or monkeys) (see, e.g., U.S. Patent No. 4,761,471 and U.S. Patent No. 4,789. 732); in vitro assays to quantify the activity of a BMP to induce proliferation of in vitro cultured cartilage cells (see, for example, U.S. Patent No. 4,795,804); in vitro assays to quantify the activity of a BMP to induce alkaline phosphatase activity in in vitro cultured muscle cells (eg, C2C12 cells, ATCC No. CRL-1772) or bone marrow stromal cells (eg, murine cells W-20, ATCC No. CRL-2623) (see, for example, U.S. Patent No. 6,593,109; Ruppert et al., Eur J Biochem 1996; 237:295-302; Kirsch et al., EMBO J, 2000; 19:3314-3324; Vallejo et al., J Biotech, 2002; 94:185-194; Peel et al., J Craniofacial Surg., 2003; 14:284-291; and Hu et al., Growth Factors , 2004; 22:29-33); in vitro assays to quantify the activity of a BMP to induce FGF receptor 2 (FGFR3) expression in cultured mesenchymal progenitor cell lines (eg, murine C3H10T1-2 cells) (see, for example, Vallejo et al. J Biotech 2002 ;94:185-194); in vitro assays to quantify the activity of a BMP to induce proteoglycan synthesis in chicken foot bud cells (see, for example, Ruppert et al., Eur J Biochem 1996; 237:295-302); and in vitro assays to quantify the activity of a BMP to induce osteocalcin treatment in bone marrow stromal cells (e.g., murine W-20 cells; ATCC No. CRL-2623) (see, e.g., U.S. Patent No. 6,593,109).

Assays for measurement of BMP binding and release

[00091] Various assays can be used to measure the binding and release of recombinant BMP from a carrier. For example, the amount of recombinant BMP protein can be quantified by any of the techniques well known in the art, including dot blots, immunoassay (e.g., enzyme-linked immunosorbent assays, ELISA), measuring the increase in radioactivity present in the BMP buffer. release when the bioimplant incorporates radiolabeled BMP and chromatography (eg, high-pressure liquid chromatography, HPLC, and ion-exchange chromatography).

[00092] Such methods are well known in the art (for example, see, Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press. 1999; Gosling, ed., Immunoassays: A Practical Approach, Oxford University Press. 2000; Oliver, ed., HPLC of Macromolecules: A Practical Approach., Oxford University Press, 1998; Millner, ed., High Resolution Chromatography: A Practical Approach. Oxford University Press, 1999; Hockfield et al., Selected Methods for Antibody and Nucleic Acid Probes, Cold Spring Harbor Laboratory Press, 1993; Gore, ed., Spectrophotometry and Spectrofluorimetry: A Practical Approach, Oxford University Press, 2000).

[00093] For example, protocols for radioimmunoassay analysis of BMP proteins have been described (see, for example, U.S. Patent No. 4,857,456). For example, protocols for immunostaining analysis of BMP proteins have been described (see, for example, Wang et al. Proc Natl Acad Sci USA 1990; 87:2220-2224). For example, ELISA kits for quantifying human, rat, or mouse BMP-2 protein levels are commercially available, for example, from R&D Systems (catalog #DBP200, PDBP200, or SBP200). For example, ELISA kits for quantifying human BMP-7 protein levels are commercially available, for example, from R&D Systems (catalog #DY354 or DY354E).

Kits

[00094] In one aspect, the invention provides a kit to contain the system described in this application. In one embodiment, the kit comprises the components needed to produce the delivery vehicle and carrier as well as the necessary growth factors. That is, the kit of the invention would comprise the components necessary to produce the delivery vehicle and the carrier as well as at least one growth factor that is associated, or later will be associated with the delivery vehicle and at least one growth factor associated with the carrier.

[00095] The kit preferably comprises a container comprising the carrier into which it can be loaded or coated with associated growth factor(s).

[00096] Preferably, the delivery vehicle and any associated growth factor(s) are kept in separate containers, which can be combined at the time of use. This would be particularly preferred in cases where the delivery vehicle may comprise a liquid or a gel. In such a case, where the delivery vehicle comprises both the associated growth factor(s) as a liquid or gel, the associated growth factor(s) may be kept in a separate container as a lyophilized powder. At the time of use, the growth factor(s), in powder form, may be combined with the liquid or gel delivery vehicle.

[00097] In a preferred embodiment, the kit of the invention would comprise at least three containers of each of the following: 1) the delivery vehicle component; 2) at least one first growth factor (ie, the growth factor(s) associated with the delivery vehicle); and, 3) the carrier and at least one second growth factor (ie, the growth factor(s) associated with the carrier). In use, at least one first growth factor, in powder form, is combined with the delivery vehicle in liquid or gel form and the mixture then applied to the carrier for which at least one second growth factor has been pre-mixed. loaded.

[00098] In another preferred embodiment, the kit of the invention would comprise at least two containers each comprising the following: 1) the delivery vehicle component; and 2) the calcium sulfate carrier and at least one growth factor associated with the carrier and one other growth factor that is not associated with the carrier. In use, the delivery vehicle in liquid or gel form is applied to the carrier and associated with the GF (bound or loaded on the GF) and growth factor that is not associated with the carrier ("free" GF). The free GF is then included in the delivery vehicle.

[00099] In a preferred embodiment, the carrier is comprised of a mixture of calcium phosphate and calcium sulfate particles. In particularly preferred embodiments of the present invention, the carrier is comprised of a mixture of calcium phosphate and calcium sulfate particles in approximately a 1:1 or 2:1 ratio. Preferably, the carrier is coated with at least one growth factor.

[000100] In yet another preferred embodiment, the kit of the invention would comprise at least three containers of each of the following: 1) the component of the delivery vehicle; 2) at least one first growth factor (ie, the growth factor(s) associated with the delivery vehicle); and, 3) the calcium sulfate carrier and at least one second growth factor (ie, the growth factor(s) associated with the carrier). In use, at least one first growth factor, in powder form, is combined with the delivery vehicle in liquid or gel form and the mixture then applied to the carrier for which at least one second growth factor has been pre-mixed. loaded.

[000101] In a preferred embodiment, the carrier is comprised of a mixture of calcium phosphate and calcium sulfate particles. In particularly preferred embodiments of the present invention, the carrier is comprised of a mixture of calcium phosphate and calcium sulfate particles in approximately a 1:1 or 2:1 ratio. Preferably the carrier is coated with at least one growth factor.

[000102] In one aspect, the kit of the invention may comprise any necessary reagents and/or instruments and/or instructions and/or vessels as may be necessary.

EXAMPLES

[000103] The present invention will now be described by means of the following examples. These examples illustrate the new findings by the inventors that a multiphase release profile of a growth factor such as rhBMP-2 produced by loading part of the BMP within a carrier that releases BMP with a sustained release and part of the BMP within a vehicle of delivery that releases BMP with an initial release is more effective than carriers that only produce a burst release or a sustained release. These examples also illustrate that calcium sulfate dihydrate or a mixture of calcium sulfate dihydrate and calcium phosphate can be used as a carrier for a growth factor such as BMP in improved systems, methods and compositions to increase the bioimplant potency.

[000104] As will be obvious to one skilled in the art, it is possible to place one growth factor within the carrier and a different growth factor within the delivery vehicle, resulting in different release profiles of each growth factor.

[000105] It will be understood that the examples provided in this application are intended only to illustrate the present invention and not to limit the scope of the invention in any way. Likewise, the invention is not limited to any particular preferred embodiment described in this application. Indeed, many modifications and variations of the invention may be apparent to those skilled in the art upon reading the present detailed description. The invention, therefore, should only be limited by the terms of the appended claims, together with the full scope of equivalents to which the claims are entitled.

EXAMPLE 1: Production of a sustained release composite carrier containing BMP by encapsulation in PLGA.

[000106] This example demonstrates how to form a carrier that contains rhBMP-2 and that releases the growth factor in a sustained release profile.

Materials and methods

[000107] PLGA 75/25 with an inherent viscosity of 1.33 dL/g (MW = 205,000-210,000) was purchased from Birmingham Polymers Inc. (Birmingham, AL). Tetracalcium phosphate (TTCP) was obtained from Taihei Chemical Industrial Co. (Osaka, Japan) and anhydrous dicalcium phosphate (DCPA) and dimethyl sulfoxide (DMSO) was obtained from Sigma Chemical Co. (MO, USA). Sugar particles were purchased from Tate & Lyle North America Inc. (Toronto, Canada).

[000108] Resorbable calcium phosphate particles were prepared by equimolar mixing of TTCP and DCPA with deionized distilled water (ddH2O) at 100% relative humidity for 24 h. The reagent was ground and sieved through a 45 μm sieve.

[000109] Recombinant human BMP-2 (rhBMP-2, Induce Biologics Inc) was prepared in the formulation buffer (1.5 mg/mL, pH 4.5; 5 mM glutamic acid, 2.5% glycine, 0 sucrose, 5% and 0.01% Tween™ 80 with ddH2O). The protein solution was added to vials containing CaP powder and stirred for at least 15 minutes. The powder was then frozen and lyophilized.

[000110] Particles with (CaP-BMP) or without (CaP)BMP were then used to produce particulate CaP-PLGA scaffold blocks by phase reversal/particle leaching as follows: PLGA was dissolved in DMSO at a concentration of 11.5% (w/v). To this solution, CaP and CaP-BMP particles were thoroughly mixed according to a CaP/PLGA ratio of 2:1 (w/w). Sugar crystals with size ranges of 0.85-1.18 mm were dispersed in CaP/PLGA and the mixture was solidified at -18°C in a mold. PLGA was precipitated and sugar crystals removed by immersion in three modifications of ddH2O.

[000111] A layer of hydroxyapatite was deposited on and along the frameworks of macroporous compounds as follows: dry cylinders of PLGA/CaP, measuring 2 mm in diameter and 2 mm in length, were pre-moistened in 70% ethanol and immersed in 60 ml PBS 3x over a period of 9 days at 37°C. SBF was prepared as follows: to 1.8 L of ddH2O under vigorous stirring the following salts were added sequentially 29.711 g of NaCl, 2.206 g of CaCl2-2H2O, 10 ml of 1 M HCl, 0.852 of Na2HPO4. The final volume was brought to 2 L. The PBS solution was changed daily. Following coating, the 3PCC samples were rinsed in ddH2O and air dried.

[000112] This resulted in the formation of a macroporous compound carrier (3PS) that is capable of releasing rhBMP-2 with a sustained release profile for at least seven days. These results are illustrated in Figure 1.

EXAMPLE 2: Production of a sustained release carrier containing BMP by encapsulation in a calcium phosphate cement

[000113] This example demonstrates how to form a calcium phosphate cement (CPC) carrier that contains rhBMP-2 having a sustained release profile.

Materials and methods

[000114] Tetracalcium phosphate (TTCP) was obtained from Taihei Chemical Industrial Co. (Osaka, Japan) and anhydrous dicalcium phosphate (DCPA) was obtained from Sigma Chemical Co. Macroporous biphasic calcium phosphate granules (Eclipse) were purchased from Citagenix (Laval Qc, Canada). Recombinant human BMP-2 (rhBMP-2, Induce Biologics Inc) was prepared in formulation buffer (1.5 mg/mL, pH 4.5; 5 mM glutamic acid, 2.5% glycine, 0.5% sucrose and 0.01% Tween™ 80 with ddH2O).

[000115] Resorbable calcium phosphate cement particles were prepared by equimolar mixing of TTCP and DCPA with the rhBMP-2 solution. The reagent was ground and sieved through a 300 and 100 μm sieve and particles between 100 and 300 μm were retained.

[000116] This resulted in the formation of calcium phosphate cement carrier particles into which rhBMP-2 was incorporated. After implantation into an animal, BMP is released in a sustained manner over a period of at least several weeks.

[000117] To produce a CPC-based sustained-release carrier that also acted as macroporous carrier CPC particles (0.1 to 0.3 mm), macroporous calcium phosphate granules (1-2 mm) were mixed in a ratio of 1 :1 (w/w).

EXAMPLE 3: Production of a BMP-containing sustained-release carrier by using a coating that binds to BMP.

[000118] This example demonstrates how to form a carrier that has a sustained release profile by applying a binding coating to the BMP. Such a method is to coat a carrier with an antibody or BMP-binding protein as described in our Copending U.S. Patent Application No. 13/002444 (the entire contents of which are incorporated into this application by reference).

Materials and methods

[000119] Polyclonal purified rabbit anti-human BMP-2 antibody was purchased from Cell Sciences, (Canton MA., Cat #PA0025). Macroporous biphasic calcium phosphate (BCP) granules (Eclipse) were purchased from Citagenix (Laval, Qc, Canada).

[000120] Sterile BCP granules were weighed in a biosafety chamber and placed in sterile TPP tubes (Mandel Scientific, Guelph ON, Canada). The antibody solution was diluted in phosphate buffered saline to a final concentration of 150, 300 and 600 ng of antibody in 1mL of PBS, filter sterilized and applied to the carrier in a 1:1 v/v ratio. Samples were stirred for at least 15 minutes at room temperature before being frozen and lyophilized. The BMP solution was then applied to the granules, allowed to soak for 15 minutes at room temperature, then frozen and re-lyophilized.

[000121] This resulted in the formation of antibody-coated BCP granules that bound and slowly released rhBMP-2 in a sustained manner.

[000122] The amount of rhBMP-2 that can be bound can be increased by increasing the amount of antibody used. The release rate can be increased by using antibodies with lower affinity or avidity.

EXAMPLE 4: Production of a delivery vehicle containing BMP using F127

[000123] This example demonstrates how to prepare a delivery vehicle containing rhBMP-2 using F127.

Materials and methods

[000124] The poloxamer was prepared as follows: 100 mL of distilled water was cooled to 4°C and various amounts of poloxamer 407 were added slowly with stirring over a period of several hours, until all the solid dry aggregate was dissolved producing a final solution ranging between 12 and 33%. The poloxamer solution was then sterilized in an autoclave (121°C, 20 minutes, 206.84 kPa). Following sterilization, the poloxamer solution was kept at 4°C until use.

[000125] Lyophilized recombinant human BMP-2 powder (rhBMP-2, Induce Biologics Inc) was added to the poloxamer solution and was slowly mixed.

[000126] Alternatively, rhBMP-2 was added from the solution (1 mg/mL, pH 4.5; 5 mM glutamic acid, 2.5% glycine, 0.5% sucrose and 0.01% Tween 80) in a ratio 1/10 or 1/20 (v/v).

[000127] This resulted in the formation of a delivery vehicle that released more than 80% of the rhBMP-2 during the first two days (as illustrated in Figure 2).

EXAMPLE 5: Production of a bioimplant with a multiphase release profile

[000128] This example demonstrates how to form a 3PS-F127 bioimplant containing rhBMP-2 that releases rhBMP-2 with a multiphase release profile.

Materials and methods

[000129] The 3PS carrier (as described in Example 1) containing 0, 4.55 or 9.1 μg of rhBMP-2 per 5 mg of the carrier was prepared and stored in Eppendorf tubes. A delivery vehicle containing 0, 4.55 or 9.1 µg of rhBMP-2 in 45.5 µl of F127 (prepared as described in Example 4) was stored in Eppendorf tubes at 4°C. Immediately before use, the F127 was pipetted into the 3PS carrier and the carrier was mixed in the delivery vehicle.

[000130] This 3PS-F127 bioimplant was then used to measure in vitro BMP release and in vivo bone formation activity as described below.

[000131] The vehicle to delivery vehicle ratios can be varied to produce the gel (1:1 v:v ratio) or putties (2:1 v:v ratio). Further, the proportion of BMP to vehicle or the particle size of the vehicle can be varied to alter the sustained release profile. Finally the amount of rhBMP-2 in the carrier and delivery vehicle can be varied to change the amount of rhBMP-2 initially released during the first few hours compared to the amount released over the next few weeks.

EXAMPLE 6: An in vitro assay for release of BMPs from bioimplants.

[000132] This example describes how to measure the release of rhBMP-2 from various bioimplants described in Examples 1 to 5.

Materials & Methods

[000133] Bioimplants containing the known amounts of rhBMP-2 prepared as in Examples 1 to 5 were transferred into Eppendorf tubes. The total amount of rhBMP-2 used was 9.1 µg of rhBMP-2 per 5 mg of carrier and 45.5 µl of F127, or 20 µg of rhBMP-2 for 10 mg of carrier at 100 µl of F127.

[000134] The samples were then incubated while shaking with a 1 ml release buffer solution comprising phosphate buffered saline (PBS) + 1% BSA at 37°C. The buffer was removed and replaced with fresh release buffer after various times (e.g. 1, 2, 5, 7 and 10 days) and the collected solutions were provided in 1.5 mL vials at -20°C for further analysis .

[000135] The amount of BMP-2 released in the buffer was measured using a commercial ELISA (Quantikine™ hBMP-2 ELISA, RnD Systems). The ELISA was performed according to the manufacturer's instructions.

Results

[000136] No BMP was detectable in the release buffer collected from any of the bioimplants that had not been loaded with BMP. Carrier samples that had been loaded with rhBMP-2 demonstrated a sustained release of rhBMP-2 over the study period, while samples in the delivery vehicle alone were released in an "initial release profile".

[000137] When the carrier and delivery vehicle were combined, various release profiles were obtained depending on which component the BMP was loaded. When 100% rhBMP-2 (9.1 µg) was loaded into the 3PS carrier (5 mg) which was then mixed with 33% F127 (45.5 µl), the BMP release profile matched the sustained model , where the amount of BMP released during the first 2 days was 5 ng, between days 3 and 5 it was 8 ng and between days 5 and 7 it was 10 ng (Figure 3; 100-0).

[000138] When 100% rhBMP-2 (9.1 μg) was loaded into 33% F127 (45.5 μl) and then mixed with the 3PS carrier (5 mg) which had no BMP in it, the BMP was released where the amount of BMP released during the first day 1 was 2363 ng, over the second day it was 381 ng and then 12 ng on the third day (Figure 3; 0-100).

[000139] When BMP was distributed between the carrier and delivery vehicle, the bioimplant demonstrated a biphasic release profile, with an initial intermediate release followed by sustained release of BMP (Figure 3; 50-50).

EXAMPLE 7: An in vitro assay to test the activity of released BMPs.

[000140] This example describes how to determine whether the rhBMP-2 released from bioimplants retains its activity. To demonstrate that the released rhBMP is biologically active, responsive cells can be cultured with the released and their response to the growth factor measured. Such assays are known in the art (see Peel et al., J. Craniofac. Surg. 2003, 14:284-291).

Materials & Methods

[000141] Materials with or without rhBMP-2 as described in Examples 1 to 5 were prepared. Releases were prepared as described in Example 3 except that the buffer was alpha minimal essential medium with 15% fetal bovine serum and antibiotics (aMEM+15%FBS+AB)

[000142] C2C12 cells were seeded in 24-well tissue culture plates at 0.5x105 cells/ml, 1ml alpha MEM+15% FBS per well. After various periods between 24 and 72 hours, the media were removed and several releases were applied. Negative controls included C2C12 cells cultured with fresh aMEM+15%FBS+AB. Positive controls included C2C12 cells incubated with aMEM+15%FBS+AB containing 25, 50, and 100 ng/ml rhBMP-2. After 48 hours, the cells were lysed in 1 ml of cell lysis buffer (Cellytic Sigma Aldrich) and the alkaline phosphatase (ALP) activity of the cell lysates measured using the para-nitrophenol phosphate assay (Sigma Aldrich). The cellular protein content of the lysates was measured using Coomassie Plus Reagent (Fisher) and was used to normalize ALP activity to the number of cells in each of the wells.

[000143] Generally, to determine if there was any loss in BMP activity when associated with the carrier or delivery vehicle, a standard activity curve of ALP/PTN results from patterns of rhBMP-2 that did not associate with the carrier or delivery vehicle is determined. The concentration of active rhBMP-2 in the releases can be determined from this standard curve and which is expressed as a percentage of the total amount of rhBMP-2 present in the releases as determined by ELISA.

EXAMPLE 8: Evaluation of osteoinductive activity of multiphase BMP implants.

[000144] This example describes how to determine the osteoinductive activity of bioimplants containing BMP in vivo. To assess the ability of bioimplants to induce bone formation, the mouse muscle pocket assay was used. In this model, the bioimplant is placed in a muscle pocket made in the hind limbs of the mouse and the size of the induced bone formed is proportional to the amount of BMP tested. Such assays are known in the art (for example, see Barr et al., Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod., 2010; 109:531-40.)

Materials and methods

[000145] The bioimplants were prepared as described in Examples 1 and 5. Under anesthesia, bilateral pouches were made in the thigh muscles of the hind limbs of male CD-1 mice aged 37-42 days, by direct dissection. The bioimplants were then placed into sterile gelatin capsules that had been placed in the muscle pocket. The muscle was brought together and the skin closed with Mitchel clips.

[000146] The animals were euthanized on day 28 post-op. Hind limbs were collected and fixed with 10% buffered formalin. Following fixation, specimens were imaged using a microCT scanner (General Electric Healthcare eXplore™ Locus SP). The samples were scanned and reconstructed using the manufacturer's program at a resolution of 59 µm. Following image reconstruction, a region of interest (ROI) was determined. This area encompassed all areas containing bone induced by the bioimplant. These can be easily distinguished from skeletal bones based on position and density.

[000147] In order to analyze the quantity and quality of bone within the ROI, the mCT image voxels were segmented into bone and non-bone phases. Segmentation was achieved by determining a threshold value for the voxel grayscale at which the voxel was counted as bone. Total volume (TV), bone volume (BV), total volume mineral density (TV-MD), bone volume mineral density (BV-MD), total volume mineral content (TV-MC), Bone volume mineral content (BV-MC) and bone volume fraction (BVF) from ROI were determined for each sample. Values were adjusted for the presence of calcium due to the carrier using an upper threshold value that only selected the carrier and subtracting it from the values obtained using a lower threshold that included the carrier plus new bone (see Humber et al., Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology and Endodontology. 2010. 109:372-384).

[000148] Following microCT analysis, the specimens were placed in branching resin or decalcified in formic acid and embedded in wax. Resin-embedded samples were evaluated by backscatter SEM while wax-embedded samples were cut and stained with hematoxylin and eosin (H&E) and examined by light microscopy to assess the types of tissue present at the implantation site.

Results

[000149] A carrier and a delivery vehicle were combined as described in Example 5.

[000150] MicroCT analysis showed that bioimplants with all BMP within the 3PS carrier, which had a sustained BMP release profile, produced the smallest bone ossicles (Figure 4; 100-0), bioimplants with all BMP within the F127 delivery vehicle, which had a burst BMP release profile, produced intermediate size ossicles (Figure 4; 0-100), whereas bioimplants with 50% BMP loaded in the carrier and 50% loaded in the delivery vehicle , which had a multiphase BMP release profile, produced the largest bone ossicles (Figure 4; 50-50).

[000151] SEM backscatter showed that, by 28 days, bone had formed along the bioimplant and towards the calcium phosphate particulate that had been incorporated into the PLGA (Figure 5). Histology confirmed that the formed tissue was bone (Figure 6).

EXAMPLE 9: An in vivo assay for the release of BMPs from bioimplants.

[000152] This example describes how to measure rhBMP-2 release from various bioimplants described in Examples 1, 2, 3, 4 or 5 after implantation in an animal. Methods for doing this are well known in the art. For example, see Uludag et al. J Biomed Mater Res, 46, 193-202, 1999.

Materials & Methods

[000153] Recombinant hBMP-2 is radiolabeled with Iodine 125 (I125) by Perkin Elmer. Radiolabeled (active) rhBMP-2 is mixed with unlabeled (non-active) rhBMP-2 to produce a 1:100 active non-active mixture.

[000154] Bio-implants containing known amounts of rhBMP-2 are prepared as in Examples 1 to 5. These bio-implants are then implanted in animals as described in Example 8. At various times, the animals are sacrificed and the implant site is dissected. The dissected tissue is then weighed, and the amount of radioactivity determined using a gamma counter.

[000155] To determine if the counts are associated with the protein, the tissue is homogenized in 0.5 mL of PBS + 0.5% BSA. Two mL of ice-cold 10% trichloroacetic acid is added to the homogenate and then it is kept for at least 1 hour at 4°C. The homogenate is then centrifuged and the supernatant removed. The radioactivity of the precipitate is then measured using a gamma counter.

[000156] The radioactivity associated with implants is corrected for decay and the total amount of BMP remaining in the implant is estimated.

EXAMPLE 10: Production of a carrier with a short sustained release profile.

[000157] This example describes ways to produce a carrier_that releases a growth factor with a short sustained release profile.

Materials & Methods

[000158] Macroporous biphasic calcium phosphate (BCP) granules (Eclipse) were purchased from Citagenix (Laval, Qc, Canada). Recombinant human BMP-2 (rhBMP-2, Induce Biologics Inc) was prepared in formulation buffer (1.5 mg/mL, pH 4.5; 5 mM glutamic acid, 2.5% glycine, 0.5% sucrose and 0.01% Tween™ 80 with ddH2O).

[000159] The sterile rhBMP-2 solution was incubated with sterile BCP granules at a ratio of 9.1 μg per 5 mg or 4.55 μg per 5 mg (BMP per BCP) for 15 minutes under agitation. Samples were then frozen and aseptically lyophilized.

[000160] Following lyophilization, the carriers were weighed in 5 mg aliquots and placed in sterile Eppendorf tubes. Some tubes had 33% F127 (45.5 µl added). The BMP release profile was then determined as described in Example 6.

Results

[000161] Carriers that were not coated with F127 (BCP) showed a burst release profile with greater amounts of BMP released over the first day and then amounts of BMP released decreasing at each subsequent time point. Mixing BCP into F127 (BCP-Pol) resulted in a short sustained release profile where a similar amount of BMP was collected each day for the first 4 days (Figure 7).

EXAMPLE 11: Changing the carrier's sustained release profile.

[000162] This example describes a means of changing the release profile of a vehicle.

Materials & Methods

[000163] PLGA with different inherent viscosities and molecular weights were purchased from Birmingham Polymers Inc. (Birmingham, AL). Carriers were then made using these PLGAs as described in Example 1. The BMP release profile of these carriers was determined according to the method of Example 6.

Results

[000164] All carriers produced sustained release profiles. However, the amount of BMP released differed depending on the viscosity/molecular weight of the PLGA used. Carriers caused low-viscosity (Pol-1) PLGA to release more rhBMP-2 than those using high-viscosity (Pol-2) PLGA over the 12-week study period (Figure 8).

EXAMPLE 12: Changing the distribution of bound and unbound proteins during lyophilization.

[000165] This example describes a means of changing the distribution of a protein between bound to the carrier particles and unbound lyophilized by varying the volume of the lyophilized solution but keeping the total protein and the fixed content of the carrier. This means allowing distribution of the protein between carrier-associated and delivery vehicle-associated protein if a delivery vehicle is later added to the lyophilization vessel.

Materials and methods

[000166] Experimental design: To test the effect that varying the volume of protein buffer added to the carrier before lyophilization has on the distribution of the lyophilized material, the ratio of carrier volume to liquid protein was varied and the total amount of protein (albumin bovine serum, "BSA") and carrier was set at 1 mg and 400 mg, respectively.

[000167] Based on the above criteria, the study was designed as shown in Table 1, in which the volume of added protein solution (i.e., 2.0, 1.5, 1.0, 0.75 and 0.5 mL /vial) and the concentration of added protein (ie, 0.5, 0.67, 1.0, 1.33, and 2.0 mg/mL) were varied. Table 1: Experimental design.

[000168] Sample preparation: BSA was prepared in formulation buffer (FB) at 2 mg/mL; 1.5mg/ml; 1mg/mL; 0.75 mg/mL 0.5 mg/mL and 0 mg/mL. 400 mg of carrier was placed in each vial containing BSA and FB. Several volumes of the protein solution were placed in each vial in the proportions given in Table 1 and the vials were held at room temperature for 30 minutes. The vials were then frozen and lyophilized. Following lyophilization, each vial was examined and the appearance of the protein lyophilisate was categorized and photographed (FIG. 9 A-C).

[000169] Grading: Protein lyophilisate distribution was graded between 0 and 4, with 0 representing no clear visible lyophilizate particles and 4 representing a clear separation of vehicle and protein with a visible protein lyophilisate sheet.

[000170] Protein measurements: To quantify the amount of protein bound to the carrier and the amount lyophilized separately from the granules, the lyophilized materials were transferred from the flask to a centrifuge tube and 1 mL of PBS was added to the centrifuge tube and flask . Containers were centrifuged and the rinse solution was collected and centrifuged. Supernatants were analyzed for protein content using the protein Coomassie-Plus assay according to the manufacturer's instructions. The amount of bound protein was calculated by subtracting the amounts of protein released from granules and vial from the amount of protein loaded (1000μg).

Results

[000171] There was a significant difference in the distribution of the lyophilisate between group 1, which had the lowest protein concentration (ie, 0.5 mg/mL) and groups 3 (1.0 mg/mL), 4 (1 .33 mg/mL) and 5 (2.0 mg/mL) (Table 2). Samples in group 1 had some protein lyophilisate visible on the walls of the vial (FIG. 9A). No lyophilisate was visible among the carrier particles in the group 1 samples. In the group 3 samples, some protein lyophilisate was visible on the glass walls of the vial. However, large chunks of the protein lyophilisate that look like snow-white or translucent flakes were also visible between the carrier particles in the group 3 samples (FIG. 9B). In samples from group 5, distinct edges of protein formed on the surface of the glass vial (FIG. 9C). These protein borders projected pieces of the protein lyophilisate. Chunks were also seen to be between the carrier particles in the group 5 samples.Table 2: Distribution of protein lyophilisate bound and unbound to granules.

[000172] Group 1 versus Group 5, Group 1 versus Group 4, and Group 1 versus Group 3 were all significantly different (Table 2).

* Ligada foi calculada subtraindo as quantidades da proteína liberada dos grânulos e o frasco da quantidade carregada (1000 μg).[000173] The measurement of bound protein indicated that there were significant differences between the amounts of bound protein based on the volume of solution added before lyophilization. Specifically, the amount of bound protein changed from 68% to 39% when the volume of solution used to deliver 1 mg of protein was changed from 0.5 mL to 2 mL per 400 mg of carrier (Table 3, see Protein bound in groups 1 and 5, respectively).Table 3: Protein measurements

* Bound was calculated by subtracting the amounts of protein released from the granules and vial from the amount loaded (1000 μg).

[000174] Post Hoc analysis of the bound protein group indicated that there were significant differences between: group 1 and groups 3, 4, and 5; group 2 and groups 4 and 5; and group 3 and group 5 (Table 4). Table 4: Comparisons for bound protein.

Example 13: Effect of varying carrier and amount of P407 on BMP release.

[000175] This example describes a means to vary the BMP release profile of the carrier by varying the amount of delivery vehicle and carrier used.

Materials and methods

[000176] Experimental design: To test the effect that varying the amount of delivery vehicle and carrier has on the BMP release profile, the amount of carrier (biphasic calcium phosphate BCP) and delivery vehicle (P407 gel 33 %) the amount was varied in bioimplants where the amount of BMP added to the carrier particles was fixed at 40μg/sample and BMP was lyophilized to the carrier granules. The study design is further described in Table 5. Table 5: Study design.

[000177] Preparation of Materials: Sterile macroporous BCP granules (diameter 0.5-1mm) comprising e-tricalcium phosphate 80%, Hydroxyapatite 20% were purchased from Citagenix Inc. (Laval, QC). BMP-2 (1mg/ml) was prepared by Induce Biologics Inc. A 33% poloxamer 407 (P407) gel was prepared by adding 33 g of poloxamer 407 (BASF) to cold water. The solution was then sterilized by autoclaving. The poloxamer gel was kept at 2-8°C after sterilization.

[000178] BMP was lyophilized for the carriers as follows. The required amount of carrier was weighed and placed in a sterile Eppendorf tube. The desired amount of BMP-2 was added to the carrier and held at room temperature for 30 minutes before freezing. Once frozen, the Eppendorf tubes were placed in a benchtop lyophilizer and lyophilized overnight. All procedures were performed aseptically to maintain sterility.

[000179] Release of BMP: The lyophilized samples were weighed and placed in Eppendorf tubes to which the P407 gel was added and allowed to soak for 20 minutes. Next, 1 mL of PBS + 0.1% BSA was added to each tube which was then placed on a shaker in a 37°C incubator and gently shaken. At each collection time point (days 1, 2, 3, 4, 7) the tubes were removed, centrifuged and PBS+BSA removed and fresh PBS+BSA added. Collected PBS+BSA was then stored frozen until analyzed.

[000180] Analytical methods: The amount of BMP released in the solutions was determined using a BMP-2 ELISA (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions

[000181] Statistical analysis: Data were tested for normality and equal variance. Normally distributed data with equal variance were tested for significant differences using 2-Way ANOVA (Vehicle and P407 were used as the factors). All other data were tested using ANOVA on RANKs. Post Hoc testing was performed on all pairs using the Student-Newman-Keuls Method. All statistical tests were performed using Sigma Stat v3.5.

Results

Tabela 7: Análise estatística de liberação de BMP ao longo de sete dias de bioimplantes contendo várias quantidades do gel de P407. De ANOVA de 2 Vias (valores P).

[000182] The addition of P407 reduced BMP release for up to 7 days (Table 6). This was a surprising result as it was previously reported that P407 gels were effective in slow drug release for only a matter of hours, after which time they had dispersed in the tampon. The amount of P407 affected BMP release during the first 7 days. From day 4, the amount of P407 initially used determines whether there is a difference between groups without P407 and + groups with P407 (Table 6). The amount of carrier present affected BMP release in the presence or absence of P407 from day 2 to 7, with increasing amount of carrier reducing the amount of BMP released (Table 6).Table 6: BMP release over time seven days of bioimplants containing various amounts of P407 gel and BCP particles.

Table 7: Statistical analysis of BMP release over seven days from bioimplants containing various amounts of P407 gel. 2-Way ANOVA (P values).

[000183] Day one data showed that there was significantly more BMP released from the carrier in the absence of P407 than when it was present (Table 8).

[000184] While the amount of carrier alone was not considered to impact BMP release with day 1 there was an interaction between amount of carrier and amount of P407. Specifically 20 mg of carrier, the amount of P407 gel used significantly influenced the released BMP (30 v 120 and 30 v 60) while in samples with 40 or 80 mg of carrier this was not observed (Table 8).Table 8: Table ANOVA for day one results.

[000185] Day two data showed that both the amount of carrier and the amount of P407 significantly impacted BMP release, with interactions occurring (Table 9). The amount of P407 needed for the effect was dependent on the amount of carrier. Table 9: ANOVA table for day two results.

[000186] Results from day three indicated that the amount of carrier was the primary factor affecting BMP release. However, the amount of P407 gel approached significance in several groups (Table 10). Table 10: ANOVA table for day three results

[000187] Results from day four indicated that both the amount of carrier and the P407 gel affected BMP release, although there appeared to be no interaction between the two (Table 11). There was no difference in the amount of the P407 gel as long as more than 30 µl of the gel had been used. BMP release differed between the 3 amounts of carrier used (Table 11). Table 11: ANOVA table for day four results.

[000188] Results from day seven were similar to the results from day 4 both with the amount of carrier particles and with P407 gel that affects BMP release (Table 12). At this time, however, a minimum amount of 60 μl of the P407 gel per implant had to be used. Table 12: ANOVA table for day seven results.

Discussion

[000189] Taken together, these results show that it is possible to vary the BMP release profile by varying the amount of P407 and the carrier used. These results also show that, in contrast to previous reports of using P407 for drug delivery over a period of a few hours, the use of the P407 gel in combination with the carrier results in inhibition of protein release for up to 7 days. It is contemplated in this application that, after most of the P407 has dissolved during the first several hours, a thin layer of the P407 gel could remain on the surface of the carrier, reducing the rate of protein release.

Example 14: Evaluation of in vitro protein release from different carrier particles.

[000190] The present example describes bioimplants comprising particles of dehydrated calcium sulfate (CSD) to which rhBMP-2 has been lyophilized. These bioimplants produced a greater and more consistent release of BMP over 14 days relative to bioimplants comprising the 2 types of calcium phosphate particles as the carrier.

Materials and methods

[000191] Experimental design: Three vehicles were tested: calcium sulfate dihydrate (CSD), hydroxyapatite (HAp) and biphasic calcium phosphate (BCP) in bioimplants, in which the proportion of BMP to the vehicle was 40 μg:20 mg, the ratio of carrier to delivery vehicle (i.e., P407) was 200 µl:20 mg and where BMP was lyophilized to vehicle granules. The experimental design is further shown in Table 13. Table 13: Vehicle Comparison.

[000192] Preparation of Materials: Sterile macroporous BCP granules (diameter 0.5-1 mm) comprising e-tricalcium phosphate 80%, Hydroxyapatite 20% were purchased from Citagenix Inc. (Laval, QC). Sterile CSD granules (0.5-1.2 mm) were prepared by grinding Osteoset precipitates (Wright Medical Technology Canada Ltd., Mississauga, ON) and sieving between 1.18 mm and 0.5 mm sieves. Sterile hydroxyapatite granules were obtained from Tissue Regeneration Therapeutics (Toronto, ON). BMP-2 (1 mg/ml) was prepared by Induce Biologics Inc. A 33% poloxamer 407 (P407) gel was prepared by adding 33 g of poloxamer 407 (BASF) to cold water. The solution was then sterilized by autoclaving. The poloxamer gel was kept at 2-8°C after sterilization.

[000193] Lyophilization of BMP for carrier particles: The required amount of vehicle was weighed and placed in a sterile Eppendorf tube. The desired amount of BMP-2 was added to the carrier and held at room temperature for 30 minutes before freezing. Once frozen, the Eppendorf tubes were placed in a benchtop lyophilizer and lyophilized overnight. All procedures were performed aseptically to maintain sterility.

[000194] BMP release in vitro: 80 μl of P407 gel were added to the carrier and associated with rhBMP-2 and left to soak for 20 minutes. Next, 1 mL of PBS + 0.1% BSA was added to each tube which was then placed on a shaker in a 37°C incubator and gently shaken. At each collection time point (days 1, 2, 3, 4, 7, 10 and 14) the tubes were removed, centrifuged and PBS+BSA removed and fresh PBS+BSA added. Collected PBS+BSA was then stored frozen until analyzed.

[000195] Analytical methods: The amount of BMP released in the solutions was determined using a BMP-2 ELISA (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions.

[000196] Statistical analysis: Data were tested for normality and equal variance. Normally distributed data with equal variance were tested for significant differences using ANOVA. All other data were tested using ANOVA on RANKS. Post-Hoc testing was performed for all pairs using the Student-Newman-Keuls Method. All statistical tests were performed using Sigma Stat v3.5.

Results

*somente uma única amostra foi medida, consequentemente ANOVA não foi realizado neste ponto de tempo.[000197] Post-Hoc testing indicated that calcium sulfate carrier particles released more BMP than BCP carrier particles at all time points tested (Table 14). CSD vehicle particles also released more BMP than Hap vehicle particles on day 1, day 2 and day 7. BMP release from HAp vehicle particles differed from BCP on days 7 and 10.Table 14: BMP release from various vehicle particles.

*only a single sample was measured, therefore ANOVA was not performed at this time point.

Discussion

[000198] These results show that CSD carrier particles released more BMP-2 than other carrier particles tested in total over the 14 days and at all but the 3 day time point.

EXAMPLE 15: Production of a BMP carrier having improved in vivo potency over known carriers used in multiphase BMP bioimplantation.

[000199] This example describes the evaluation of various carrier components to determine which one produces the most bone when used as part of a multiphase BMP bioimplant. In this example "improved bone growth" or "improved ability for ossicle formation" refers to an increase in size and/or density of ossicles over that of known carriers comprising the same BMP. The results of this study show that bioimplants using CSD particles, for which rhBMP-2 was lyophilized, produced larger ossicles when implanted than bioimplants containing hydroxyapatite or biphasic calcium phosphate carriers.

Materials and methods

[000200] Experimental design: To identify a carrier with relatively high bone production capacity, calcium sulfate dihydrate (CSD), hydroxyapatite (HAp) and biphasic calcium phosphate (BCP) were tested in bioimplants in which the proportion of BMP to implant volume was set at 40 µg:20 mg, the ratio of carrier to delivery vehicle (ie P407) was set at 200 µl:20 mg, and BMP was lyophilized to carrier granules. The experimental design is also shown in Table 15. Table 15: Vehicle Comparison

[000201] Preparation of Materials: Sterile macroporous BCP granules (diameter 0.5-1 mm) comprising 80% e—tricalcium phosphate, 20% hydroxyapatite was purchased from Citagenix Inc. (Laval, QC). Sterile CSD granules (0.5-1.2 mm) were prepared by grinding Osteoset precipitates (Wright Medical Technology Canada Ltd., Mississauga, ON) and sieving between 1.18 mm and 0.5 mm sieves. Sterile hydroxyapatite granules were obtained from Tissue Regeneration Therapeutics (Toronto, ON). BMP-2 (1 mg/ml) was prepared by Induce Biologics Inc. A 33% poloxamer 407 (P407) gel was prepared by adding 33 g of poloxamer 407 (BASF) to cold water. The solution was then sterilized by autoclaving. The poloxamer gel was kept at 2-8°C after sterilization. BMP was lyophilized for carriers as follows: the required amount of carrier was weighed out and placed in a sterile Eppendorf tube. The desired amount of BMP-2 was added to the carrier and held at room temperature for 30 minutes before freezing. Once frozen, the Eppendorf tubes were placed in a benchtop lyophilizer and lyophilized overnight. All procedures were performed aseptically to maintain sterility.

[000202] Surgical model: The osteoinductivity of various bioimplants was evaluated in the mouse muscle pouch assay (Barr et al. Radiol Oral Endod Oral Pathol Oral Med Oral Surg. 2010; 109(4):531-40).

[000203] The samples, where the poloxamer was to be mixed with the carrier, were prepared by pouring the carrier granules onto a sterile stainless steel tray. The poloxamer was kept on ice and an appropriate amount of the poloxamer gel was pipetted onto the carrier beads. The carrier and gel were mixed and then carefully placed in a gelatin capsule which was then placed in the muscle pouch.

[000204] Male IGS mice (approximately 22 grams) had intramuscular pockets formed in their biceps femoris muscle by direct dissection. The bioimplant was then placed in the bag. The skin was then brought together and closed using Michel clips.

[000205] The mice were monitored daily. Originally the mice were supposed to be euthanized after 28 days. However, due to some implants that formed so much bone that the junction occurred between the spine and the femur, which restricted the mice's mobility, all mice were sacrificed after 18 days. Following animal sacrifice, the hind limbs were dissected and fixed using neutral buffered formalin.

[000206] Analytical methods: The amount of bone formed by the bioimplants was determined by micro CT. Appropriate values were adjusted for the presence of residual carrier calcium as previously described ( Humber et al. Oral Surg. Oral Pathol Oral Med. Oral Radiol. Oral Endod. 2010 Mar; 109(3):372-84 ). Briefly, the region where the implant had been placed was imaged using a General Electric Healthcare eXplore™ Locus SP microCT scanner. The residual carrier and any new mass that formed around the implant in the muscle (collectively called the ossicle) were outlined every 10 slices to define the region of interest (ROI).

[000207] The carrier material was denser than the new bone. Therefore, it was possible to determine threshold values for new bone and carrier separately by imaging multiple samples from each group and taking an average of the grayscale values. For standardization purposes, the lowest carrier threshold value obtained for a material (ie, CSD) was used for all carriers (ie, 1835). Similarly, a single value of new bone was used (ie, 555).

Tabela 17: Parâmetros calculados (Limite inferior-limite superior).

[000208] Analyzes were performed using the 2 threshold values (ie 1835 and 555). The upper threshold distinguished bone and soft tissue carrier, while the lower threshold distinguished bone + soft tissue carrier. By subtracting the upper limit values from the lower limit values, bone only values were determined. Seven different parameters were measured using microCT. Table 16 describes the parameters obtained directly from the microCT and any thresholds that impacted the result. Table 17 describes the derived parameters and how they are calculated. Table 16: Reported parameters provided by microCT.

Table 17: Calculated parameters (Lower limit-upper limit).

[000209] The two measurements used to determine osteoinductive activity were total volume (TV) and adjusted bone volume (aBV).

[000210] Histology: After micro CT analysis, the samples were decalcified and processed for optical microscopy.

[000211] Statistical analysis: MicroCT parameters were tested for normality and equal variance. Normally distributed data with equal variance were tested for significant differences using ANOVA. All other data were tested using ANOVA on RANKS. Post-Hoc testing was performed on all pairs using the Student-Newman-Keuls Method. All statistical tests were performed using Sigma Stat v3.5.

Results

[000212] Vehicle Comparison: MicroCT results indicated that CSD carriers produced larger ossicles than any calcium phosphate-based carriers by total volume (Table 18). There was also a tendency for the CSD ossicles to contain more new bone than any carriers containing calcium phosphate (Table 19).Table 18: Total volume (mm3)

[000213] Post Hoc Test: All multiple pairwise comparison procedures (Student-Newman-Keuls method). CSD against HAp (P<0.05); CSD versus BCP (P<0.05); HAp versus BCP (no significant difference). Table 19: Adjusted Bone Volume (mm3)

[000214] Histology: Histological evaluation indicated that for all bioimplants, the ossicles comprised primarily a shell of bone surrounding a mixture of bone, cartilage, and marrow tissue. There was no sign of inflammation in any of the implants.

[000215] Residual calcium phosphate granules were visible in bioimplants containing BCP or HAp while CSD appears to be rapidly resorbed with only a few CAS granules seen.

[000216] These results show that when BMP-2 was lyophilized for CSD carriers the CSD-P407 bioimplant produced larger ossicles containing more bone than other vehicles for which BMP-2 was lyophilized.

EXAMPLE 16: Production of a bioimplant having increased potency by delivering BMP to both the carrier and the delivery vehicle compared to bioimplants where the BMP was only in the vehicle.

[000217] This example describes the evaluation of calcium phosphate and calcium sulfate individually and as a mixture, as BMP carriers that could improve bone production relative to ACS in mouse muscle pouch assays, in which a preferred ratio of BMP in the carrier to delivery vehicle (i.e., P407) and a preferred ratio of calcium sulfate to biphasic calcium phosphate is determined.

Materials and methods

[000218] Experimental design: To identify a carrier with relatively high capacity for bone production, mixtures of dehydrated calcium sulfate (CSD) and biphasic calcium phosphate (BCP) in proportions of 1:0, 3:1, 1:1 and 0:1 were tested. Furthermore, the ratio of BMP in the carrier versus the F127 delivery vehicle was varied such that the ratios of 100:0, 90:10 and 70:30 CSD:BCP were tested. Each variable was tested in a bioimplant where the ratio of BMP to implant volume was 40 µg: ~50 µl, based on a target of <1 mg/cm3 and where the ratio of carrier to F127 was 30:45. The experimental design is also shown in Table 20. Table 20: Experimental design.

[000219] Preparation of Materials: Sterile macroporous BCP granules (diameter 0.5-1 mm) comprising e-tricalcium phosphate 80%, Hydroxyapatite 20% were purchased from Citagenix Inc. (Laval, QC). Sterile CSD granules (0.5-1.2 mm) were prepared by grinding Osteoset precipitates (Wright Medical Technology Canada Ltd., Mississauga, ON) and sieving between 1.18 mm and 0.5 mm sieves.

[000220] The Infuse® kit was purchased from Medtronic of Canada Ltd. of BMP-2 Infuse was prepared by adding water for injection to the lyophilized rhBMP-2 powder in the Infuse® kit. The ACS sponge was cut into approximately 5x5 mm pieces and placed in Eppendorf capsules.

[000221] Inducing BMP-2 (1 mg/ml) was prepared by Induce Biologics Inc. A 33% poloxamer 407 (P407) gel was prepared by adding 33 g of poloxamer 407 (BASF) to cold water. The solution was then sterilized by autoclaving. The poloxamer gel was kept at 2-8°C after sterilization.

[000222] BMP was lyophilized for the carriers as follows: The required amount of carrier was weighed and placed in a sterile Eppendorf tube. The desired amount of BMP-2 was added to the vehicle and held at room temperature for 30 minutes before freezing. Once frozen, the Eppendorf tubes were placed in a benchtop lyophilizer and lyophilized overnight. All procedures were performed aseptically to maintain sterility. BMP-P407 samples were prepared in bulk in sterile Eppendorf tubes by adding BMP-2 to BMP at the desired concentration. At the time of surgery, the appropriate amount of P407 was pipetted out of the tube.

[000223] Surgical model: As shown in Example 15.

[000224] Analytical methods: micro CT and histology analyzes were as presented in Example 15.

[000225] Statistical analysis: As ACS alone did not form ossicles that can be measured it was not included in any statistical analysis.

[000226] The microCT parameters were tested for normality and equal variance. Normally distributed data with equal variance were tested for significant differences using ANOVA. All other data were tested using ANOVA on RANKS. Post-Hoc testing was performed on all pairs using the Student-Newman-Keuls Method. All statistical tests were performed using Sigma Stat v3.5.

Results

[000227] Effect of BMP Distribution between the carrier granules and the P407 gel: When BMP was distributed between the P407 gel and the CSD granules it produced larger ossicles than when all the BMP were lyophilized for the CSD (volume of whole bone). (Group 3a > Group 2a) (Table 22).

[000228] Using 2:1 CSD-BCP granules, more bone was formed when 70% was lyophilized for the granules and 30% was in the P407 gel then when 90% was lyophilized and 10% was in the gel (total bone volume ). (Group 5a > Group 7a) (Table 22).

Tabela 22: Teste Post Hoc (comparação de grupos contendo BMP em análise de volume total). Todos os Múltiplos Procedimentos de Comparação aos Pares (Método de Student-Newman-Keuls).

[000229] Effect of using CSD granules instead of BCP: In groups with the same distribution of BMP between granules and P407 we found that using CSD granules produced larger ossicles than BCP (total bone volume) (Gp 3a> Gp 4a) (Table 20). When CDS was mixed with BCP groups with CSD of more than 50% in proportion, it formed the largest ossicles (Group 3a (100CSD) > 5a (67% CSD) > Group 7a (50% CSD) = 4a (0% of CSD) (total bone volume) (Table 22).Table 21: MicroCT, the total volume of bone produced.

Table 22: Post Hoc Test (comparison of groups containing BMP in full volume analysis). All Multiple Pair Comparison Procedures (Student-Newman-Keuls Method).

[000230] Effect of BMP Distribution between the granules and the P407 gel: When BMP was distributed between the P407 gel and the CSD granules, more bone was produced than when all the BMP were freeze-dried for CSD (bone volume adjusted). (Gp3a > Gp2a) (Table 24).

Tabela 24: Teste Post Hoc (comparação de grupos contendo BMP). Todos os múltiplos procedimentos de comparação aos pares (Método de Student-Newman-Keuls):

[000231] Effect of using CSD instead of BCP granules: In groups with the same distribution of BMP between granules and P407 we found that using CSD granules, larger ossicles were produced than BCP (adjusted bone volume) (Gp 3a> Gp 4a) (Table 24). When CDS was mixed with BCP groups with CSD of more than 50% in proportion it formed the largest ossicles (adjusted bone volume) (Gp3a (100CSD) > 5a (67% CSD) > 4a (0% CSD) (Table 24 Table 23: Adjusted Bone Volume (aBV) All multiple pairwise comparison procedures (Student-Newman-Keuls Method).

Table 24: Post Hoc test (comparison of groups containing BMP). All multiple pairwise comparison procedures (Student-Newman-Keuls Method):

[000232] Histological evaluation indicated that for all bioimplants, the ossicles comprised primarily a shell of bone surrounding a mixture of bone, cartilage, and marrow tissue. There was no sign of inflammation on any of the implants.

[000233] Residual calcium phosphate granules were visible in the Induce Bioimplants containing BCP, while calcium sulfate appears to be undergoing rapid resorption with only a few CAS granules seen. Bone was seen forming directly on and within CAS and BCP granules (FIG. 10A-B).

Discussion

[000234] The results from this study show that when BMP was distributed between being lyophilized to vehicle granules and mixed into the P407 gel, (a distribution that results in a multiphasic release of BMP) the largest ossicles with the most bone were produced than that when all the BMP were lyophilized to the granules which were later mixed with the P407 gel at the time of surgery

[000235] A 70/30 split between granules and P407 gel produced larger ossicles with more bone than a 90/10 split between P407 gel and granules.

[000236] In groups with an equal distribution of BMP between granules and P407 CSD granules produced larger ossicles with more bone than similarly graded BCP granules, despite CSD-produced ossicles having a greater surface area due to being porous ( BCP granules were solid). When CSD could be mixed with BCP bioimplants with 67% or more CSD granules produced larger ossicles than those with 50% or less CSD granules.

Claims (8)

1. Multiphase delivery system for growth factors at a treatment site, characterized in that it is a bioimplant, comprising: (a) a delivery vehicle, in the form of a liquid or a gel, comprising a first factor growth factor, the polymer being poly(ethylene glycol)-block-poly(propylene glycol)-block-pol(ethylene glycol) (poloxamer 407), the delivery vehicle being adapted to release a first growth factor in a growth profile initial release for a first period of time; and, (b) a carrier comprising a plurality of solid particles having a second growth factor on the surface thereof, the carrier being adapted to release the second growth factor in a sustained release profile over a second period of time, and being that the carrier comprises calcium sulfate dihydrate particles; wherein the carrier particles are combined with the delivery vehicle; the first and second growth factors being bone morphogenetic proteins (BMPs); and wherein the first time period comprises hours or days, the second time period comprises days or weeks, and wherein the second time period is of a longer duration than the first time period. 2. System, according to claim 1, characterized in that the carrier also comprises calcium phosphate particles, preferably with the proportion of calcium sulfate dihydrate for calcium phosphate particles being 1:1 or 2:1. 3. System, according to claim 1 or 2, characterized by the fact that the first growth factor and the second growth factor are the same. 4. System according to any one of claims 1 to 3, characterized in that the delivery vehicle comprises at least 10% of the total amount of the growth factor, and the vehicle comprises at least 50% of the total amount of the factor of growth. 5. System, according to any one of claims 1 to 4, characterized in that the proportion of delivery vehicle to carrier is 0.5:1 to 4:1 (v/v). 6. System according to any one of claims 1 to 5, characterized in that the second growth factor is applied to the carrier, and then lyophilized in the carrier. 7. System according to any one of claims 1 to 6, characterized in that the bone morphogenetic proteins are selected from among bone morphogenetic protein-2 (BMP-2) and bone morphogenetic protein-7 (BMP-7). 8. System according to any one of claims 1 to 7, characterized in that the delivery vehicle is adapted to release at least 80% of the first growth factor within a period of 72 hours.

Images